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PublicationArticle Antitumor potential of ivermectin against T-cell lymphoma-bearing hosts(Springer, 2025) Alok Shukla; Arpit Sharma; Shivani Gupta; Abha Mishra; Amit Kumar SinghIvermectin, a broad-spectrum antiparasitic agent from the ivermectin family, has shown promising anticancer potential. Originally developed for veterinary and human use against parasitic infections, ivermectin demonstrated significant antitumor effects in our study against tumor cells (Dalton’s lymphoma cells). A dose-dependent decrease in tumor cell viability was observed following 24-h treatment with ivermectin, with an IC₅₀ value calculated at 10.55 µg/mL. In comparison, the standard anticancer drug cisplatin exhibited a slightly higher cytotoxic potency, with an IC₅₀ of 8.32 µg/mL under the same treatment duration. Flow cytometric analysis revealed that ivermectin induced cell cycle arrest in the G0–G1 phase. Apoptotic tumor cell death was confirmed via Annexin V/PI staining, further supported by nuclear condensation, a hallmark of apoptosis, visualized through both confocal microscopy and flow cytometry. The apoptosis was determined to be mitochondrial-dependent, as evidenced by a decline in mitochondrial membrane potential (ΔΨm) observed through JC-1 assay. The treatment increased DAPI-positive cells and exhibited severe chromatin condensation. Additionally, cell death was validated using Acridine Orange and Propidium Iodide staining, which highlighted increased cell membrane rupture and death through apoptosis and necrosis. Mitochondrial dependent apoptosis further supported by increased ROS production upon ivermectin treatment. Moreover, In vivo, ivermectin treatment led to a substantial reduction in tumor size in tumor-bearing mice, along with normalization of spleen size, body weight, and improvement histopathology of liver. These findings collectively support the therapeutic potential of ivermectin as a repurposed anticancer agent, acting through multiple mechanisms including cell cycle arrest, ROS generation, mitochondrial dysfunction, and apoptosis. © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2025.PublicationArticle Down regulation of CD24 and HER-2/neu in breast carcinoma cells by activated human dendritic cell. Role of STAT3(2012) Sumit Kumar Hira; Partha Pratim MannaHuman dendritic cells (DCs) stimulated with cytokines and LPS down regulate the expression of proto-oncogene HER-2/neu and GPI linked protein CD24 in breast cancer cell lines. We demonstrated that naïve DC from human peripheral blood, when stimulated with IFN-γ, IL-15 or LPS reduces the expression of HER-2/neu and CD24, via activation of TNF-α. Pretreatment of tumor cells with STAT3 specific inhibitors or knocking down of STAT3 by SiRNA makes the tumor cell more susceptible to apoptosis and DC mediated inhibition of both CD24 and HER-2/neu. Thus DC could acts as an inhibitory regulator in suppressing oncogene and prevention of metastasis. © 2012 Elsevier Inc.PublicationArticle Activation of p53-dependent/-independent pathways of apoptotic cell death by chelerythrine in a murine T cell lymphoma(Informa Healthcare, 2015) Sanjay Kumar; Munendra Singh Tomar; Arbind AcharyaThe p53 tumor suppressor protein has been implicated as an activator of apoptosis. In order to investigate the effect of chelerythrine and staurosporine on the activation of p53-dependent/-independent pathways of Dalton lymphoma (DL) cell death, cells were treated with chelerythrine and staurosporine for 1 h, 3 h and 6 h, respectively. It was found that treatment with chelerythrine and staurosporine increased the expression of total-p53/phospho-53 (ser-15) significantly at protein and mRNA levels, which resulted in activation of the p53-dependent apoptotic pathway in DL cells. In addition, increased activities of cyt-c, caspase-9 and caspase-3 and degradation of DNA into fragments confirmed activation of the p53-independent apoptotic pathway in p53 knockdown RNAi-DL cells. In brief, the present study demonstrated activation of p53-dependent/-independent apoptotic pathways in DL cells. Therefore, targeting of p53-dependent/-independent apoptotic pathways may lead to the possibility of designing and developing better therapeutic regimens to treat DL and other human cancers. © 2015 Informa UK, Ltd.PublicationArticle Benzothiazole derivatives bearing amide moiety: Potential cytotoxic and apoptosis-inducing agents against cervical cancer(Lippincott Williams and Wilkins, 2016) Meenakshi Singh; Arusha Modi; Gopeshwar Narayan; Sushil K. SinghCervical cancer is a major cause of morbidity and mortality in women worldwide. In recent years, benzothiazole analogues have attracted considerable attention in anticancer research. Therefore, in this study, the earlier reported amide series of benzothiazole derivatives were investigated for their antiproliferative activity. The activity of amide derivatives was evaluated using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometric analysis, apoptosis assay, and DNA fragmentation on two human cervical cancer cell lines: SiHa and C33-A. The data reported from this investigation indicated that benzothiazole derivatives show pronounced cytotoxicity in the HPV16-positive SiHa cells compared with HPV-negative C-33A cells. The in-vitro cytotoxicity of the compounds on the HEK-293 noncancer cell line was evaluated to establish selectivity. Cells treated with benzothiazole derivatives showed prominent morphological features as evidenced by cell shrinkage, membrane blebbing, apoptotic nuclei, and DNA fragmentation. The benzothiazole derivatives show accumulation of cells in the sub-G1 and S-phase of the cell cycle in SiHa and C33-A, respectively. In addition, these derivatives exert their beneficial effect by inducing apoptosis, in the chemoprevention of cervical cancer cells, and were further ascertained using a DNA fragmentation assay. The compounds studied showed potent cytotoxic and apoptotic properties against SiHa and C33-A cancer cell lines and thus represent an excellent starting point for further optimization of therapeutically effective anticancer drugs. Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.PublicationArticle Withania somnifera Alleviates Parkinsonian Phenotypes by Inhibiting Apoptotic Pathways in Dopaminergic Neurons(Springer Science and Business Media, LLC, 2014) Jay Prakash; Shikha Chouhan; Satyndra Kumar Yadav; Susan Westfall; Sachchida Nand Rai; Surya Pratap SinghManeb (MB) and paraquat (PQ) are environmental toxins that have been experimentally used to induce selective damage of dopaminergic neurons leading to the development of Parkinson’s disease (PD). Although the mechanism of this selective neuronal toxicity in not fully understood, oxidative stress has been linked to the pathogenesis of PD. The present study investigates the mechanisms of neuroprotection elicited by Withania somnifera (Ws), a herb traditionally recognized by the Indian system of medicine, Ayurveda. An ethanolic root extract of Ws was co-treated with the MB–PQ induced mouse model of PD and was shown to significantly rescue canonical indicators of PD including compromised locomotor activity, reduced dopamine in the substantia nigra and various aspects of oxidative damage. In particular, Ws reduced the expression of iNOS, a measure of oxidative stress. Ws also significantly improved the MB + PQ mediated induction of a pro-apoptotic state by reducing Bax and inducing Bcl-2 protein expression, respectively. Finally, Ws reduced expression of the pro-inflammatory marker of astrocyte activation, GFAP. Altogether, the present study suggests that Ws treatment provides nigrostriatal dopaminergic neuroprotection against MB–PQ induced Parkinsonism by the modulation of oxidative stress and apoptotic machinery possibly accounting for the behavioural effects. © 2014, Springer Science+Business Media New York.PublicationArticle Structure-guided discovery of a novel BTK inhibitor inducing apoptosis and G1 phase arrest in tumor cells(Springer Nature, 2025) Alok Shukla; Arpit Sharma; Shivani Gupta; Shruti Sharadrao Raut; Abha Mishra; S. Hemalatha; Amit Kumar SinghBruton’s tyrosine kinase (BTK) is a pivotal component of multiple signaling pathways in hematopoietic cells and serves as a critical pharmacological target in B-cell malignancies. Despite the availability of clinically approved BTK inhibitors, therapeutic resistance and limited efficacy in certain patient populations necessitate the discovery of novel candidates. In this study, virtual high-throughput screening of the ZINC database was employed to identify potential BTK inhibitors. Compounds were prioritized based on molecular docking scores, binding patterns, and free energy calculations. ZINC000045971961 (ZINC1961) emerged as a promising lead compound, forming stable hydrogen bonds with Glu475 and Met477 key residues also targeted by the reference inhibitor Ibrutinib. Molecular dynamics simulations and MM/GBSA free energy analysis further confirmed the stability and favorable binding affinity of ZINC1961. Biological evaluation in primary tumor cells demonstrated potent cytotoxicity, with an IC50 of 80 ± 0.5 µM, and pronounced apoptosis confirmed by AO/EB/DAPI triple staining, Annexin-V/PI assay, and scanning electron microscopy. Additionally, ZINC1961 induced G1 phase cell cycle arrest, contributing to its antiproliferative effects. Collectively, these findings not only highlight ZINC1961 as a novel BTK inhibitor but also underscore the power of integrative in silico and in vitro approaches in accelerating early-stage cancer drug discovery. © The Author(s), under exclusive licence to Springer Nature Switzerland AG 2025.PublicationArticle Dysregulation of apoptotic pathway candidate genes and proteins in infertile azoospermia patients(Elsevier Inc., 2015) Deepika Jaiswal; Sameer Trivedi; Neeraj K. Agrawal; Kiran SinghObjective To dissect the role of the apoptotic pathway and its regulation in the pathogenesis of male infertility in nonobstructive azoospermia. Design Prospective study. Setting University hospital. Patient(s) Sixty-three infertile azoospermic patients with different histologic phenotypes were recruited (obstructive azoospermia, n = 16; hypospermatogenesis, n = 11; maturation arrest, n = 15; Sertoli cell only, n = 21). Intervention(s) Testicular biopsies for histopathologic and expression analysis. Main Outcome Measure(s) Expression analysis by quantitative reverse transcription-polymerase chain reaction, protein localization by immunohistochemistry and apoptotic proteome array. Result(s) Results showed significantly increased expression of proapoptotic proteins like BAX, BAD, and BAK and comparatively lowered expression of antiapoptotic BCL2 and BCLW. Immunostaining revealed increased active caspase-3 activity and more TUNEL-positive cells in different impaired phenotypes as compared with normal. In addition, significantly increased m-RNA expression of TGFB1, P53, and FASLG along with significant down-regulation of VEGFA were observed. Expression of phosphorylated P53 at the S15 position and phosphorylated RAD17 at S635 was observed in cases with spermatogenic impairment at the translational level. Conclusion(s) The results clearly indicate increased levels of apoptosis along with its other regulatory factors. The balance between pro- (BAX and BAK) and antiapoptotic (BCL2 and BCLW) genes was disturbed, which may lead to altered apoptosis. Therefore, altered regulation of apoptosis might be associated with impaired spermatogenesis, eventually leading to male infertility. © 2015 American Society for Reproductive Medicine.PublicationArticle Cyclophosphamide-induced agenesis of cerebral aqueduct resulting in hydrocephalus in mice(2007) Prakash; Gajendra Singh; Sukh Mahendra SinghThe present work was undertaken to reveal the mechanism of cerebral aqueduct agenesis found to result in hydrocephalus following intrauterine exposure to model teratogen, cyclophosphamide, in murine fetuses. A single dose of 10-mg/kg body weight cyclophosphamide was injected intaperitoneally to pregnant mice on day 10, 11 or 12 of gestation. Fetuses were collected through abdominal incision on day 18 and studied for various malformations of brain and cranium including hydrocephalus. Incomplete development and failure of canalization of the cerebral aqueduct were detected when serial sections of brain in coronal and transverse planes were studied under the microscope. Biotechnological investigations such as % DNA fragmentation, % viable cell count and cell proliferation assay were carried out on brain cells for further studies. Agenesis and non-canalization of the cerebral aqueduct resulted in increased pressure of CSF, which led to rupture of the aqueduct complicated by leakage and accumulation of CSF in brain substance forming a cavity containing CSF parallel and lateral to the unopened part of the cerebral aqueduct. Incomplete development along with non-canalization of the cerebral aqueduct resulted in blockage of CSF flow through the ventricles that manifest as internal hydrocephalus. External hydrocephalus on the other hand was detected where the CSF accumulated in the cavity formed inside the brain substance and established communication with the CSF in the subarachnoid space. Cyclophosphamide induced inhibition of mitosis and cell differentiation of ependymal cells reflecting a decreased % viable cell count and cell proliferation assay along with augmentation of apoptosis of brain cells quantified as increased % DNA fragmentation count, which were identified as the contributing factors underlying the agenesis and incomplete development of the cerebral aqueduct. The study also suggests that cell survival, proliferation, migration or differentiation of ependymal cells might have been affected, and we speculate that CSF may have an inducing role in the development and canalization of the cerebral aqueduct. © 2007 Springer-Verlag.PublicationArticle GLP 1 regulated intestinal cell’s insulin expression and self-adaptation before the onset of type 2 diabetes(Tabriz University of Medical Sciences, 2019) Shivani Srivastava; Harsh Pandey; Surya Kumar Singh; Yamini Bhusan TripathiPurpose: Basically insulin is known to be secreted by β cells of the pancreas. Recently, it has also been found to be produced and expressed by intestinal epithelial cells with the help of L cells secreting glucagon like peptide 1 (GLP 1). Here, we have studied the same intestinal insulin expression property in T2D rats. Methods: Following 2 weeks of high fat diet (HFD) consumption, we have been given a single dose of streptozotocin (STZ) (35 mg/kg bw). Rats were then sacrificed after 1, 7 and 21 days. The GLP 1 analogue, liraglutide was also given to one group of diabetic rats, upto their respective durations. Intestinal cells apoptosis were checked by tunnel assay, Incretin hormones secretion and dipeptidyl peptidase 4 (DPP-IV) activity were analyzed through ELISA and immunohistochemistry was used to determine the insulin expression of intestine at different time interval during diabetes progression. Results: As compared to 1 and 21 days, we have found minor cells apoptosis in 7 days group along with high level of GLP 1 in diabetic model. Further, these effects were enhanced by liraglutide. In response to these we have found, decreased insulin expression after 21 days and with no significant effect upto 7 days in diabetic control groups. In contrast to this, GLP-1 level and insulin expression enhances prominently after 7 days of liraglutide treatment. Conclusion: These results explain the self-adapting approach of intestinal cells against diabetes onset and insulin expression enhancing property of liraglutide under stressful conditions. This study should be continued in future for the development of intestinal insulin producing drugs, to control diabetes under irreversible β cells damage. © 2019 The Author (s).PublicationArticle Melatonin Ameliorates LPS-Induced Testicular Nitro-oxidative Stress (iNOS/TNFα) and Inflammation (NF-kB/COX-2) via Modulation of SIRT-1(Springer Science and Business Media Deutschland GmbH, 2021) Jitendra Kumar; Chandana Haldar; Rakesh VermaLipopolysaccharide (LPS) — an endotoxin that is being extensively used in laboratory to mimic microbial infection that adversely affects male fertility. This study investigated the protective effects of melatonin on LPS-induced testicular nitro-oxidative stress, inflammation, and associated damages in the testes of male golden hamsters, Mesocricetus auratus. Hamsters were administered with melatonin and LPS for 7 days. Testes of LPS treated hamsters showed degenerative changes (appearance of vacuoles, exfoliation, and depletion of germ cells in the seminiferous tubules), adverse effects on spermatogenesis (sperm count and viability), and steroidogenesis (declined serum and testicular testosterone). Furthermore, LPS treatment decreased melatonin content, melatonin receptor (MT1), and antioxidant potential (catalase and SOD), and simultaneously increased nitro-oxidative stress (CRP, nitrate, TNFα). LPS upregulated NF-kB, COX-2, and iNOS expressions to increase testicular inflammatory load that resulted in the decrease of germ cell proliferation and survival, thus culminating into germ cell apoptosis as indicated by AO-EB staining and caspase-3 expression. Administration of melatonin with LPS showed improved testicular histoarchitecture, sperm parameters, and testosterone level. Melatonin increased testicular antioxidant status (SOD, catalase) to counteract the LPS-induced testicular ROS and thus reduced testicular nitro-oxidative stress. Furthermore, melatonin treatment upregulated testicular SIRT-1 expression to inhibit LPS-induced inflammatory proteins, i.e., NF-kB/COX-2/iNOS expression. The rescue effect of melatonin was further supported by increased germ cell survival (Bcl-2), proliferation (PCNA), and declined apoptosis (caspase-3). In conclusion, our result demonstrated that melatonin rescued testes from LPS-induced testicular nitro-oxidative stress, inflammation, and associated damages by upregulation of SIRT-1. © 2021, Society for Reproductive Investigation.